A great deal is known about human platelet cells. General publications describing techniques, materials, and methods for the storage of platelets are described by Murphy et al. in "Improved Storage of Platelets for Transfusion in a New Container", Blood 60(1):194-200 (1982); by Murphy in "The Preparation and Storage of Platelets for Transfusion", Mammon, Barnhart, Lusher, and Walsh, PJD Publications, Ltd., Westbury, N.Y. (1980); by Murphy in "Platelet Transfusion", Progress in Hemostasis and Thrombosis, Vol. III, Ed. by T. Spaet, Grune and Stratton, Inc. (1976); by Murphy et al. in "Platelet Storage at 22.degree. C.: Role of Gas Transport Across Plastic Containers in Maintenance of Viability", Blood 46(2):209-218 (1975); by Kilkson, Holme, and Murphy in "Platelet Metabolism During Storage of Platelet Concentrates at 22.degree. C.", Blood 64(2):406-414 (1984); by Murphy in "Platelet Storage for Transfusion", Seminars in Hematology 22(3): 165-177 (1985); by Simon, Nelson, Carmen, and Murphy in "Extension of Platelet Concentrate Storage", Transfusion 23:207-212 (1983); by Cesar, Diminno, Alam, Silver, and Murphy in "Plasma Free Fatty Acid Metabolism During Storage of Platelet Concentrates for Transfusion", Transfusion 27(5):434-437 (1987), each of which publications is hereby incorporated by reference as if more fully set forth herein.
In order to maintain viability, platelets must generate new adenosine triphosphate (ATP) continuously to meet their energy needs. Two chemical pathways are generally available: glycolysis and oxidative phosphorylation. In glycolysis, one molecule of glucose is converted to two molecules of lactic acid generating two molecules of ATP. In oxidation, glucose, fatty acid or amino acid enters the citric acid cycle and is converted to CO2 and water. This pathway requires the presence of an adequate supply of oxygen. It is much more efficient than glycolysis, producing 36 molecules of ATP per molecule of glucose.
It has been recognized that platelets will meet their energy needs in a manner which is not necessarily consistent with their long term storage ex vivo in a viable condition. When given adequate oxygen, platelets produce most of their required ATP through oxidation, but continue to produce lactic acid through glycolysis instead of diverting all metabolized glucose through the oxidative pathway. Therefore, during storage of platelets in plasma, a glucose-containing medium, lactic acid concentrations have been found to rise approximately 2.5 mM per day. This leads to a gradual fall in pH, even in the presence of naturally occurring plasma buffers, principally sodium bicarbonate.
A considerable body of prior art exists concerning storage of platelets. Prior work has shown that the duration of platelet storage is limited by the continuing production of lactic acid by platelets. Although this provides energy for the platelets, the lactic acid produced acidifies the medium containing the platelets, which eventually destroys the cells. It is also known that fatty acids and amino acids may be used as substrates for oxidative metabolism of stored platelet cells.
In routine blood banking practice, platelet concentrates (PC) are prepared by drawing a unit of blood (about 450 ml) into a plastic bag containing an anticoagulant and then centrifuging the blood into three fractions: red cells, plasma, and platelets. The separated platelet fraction is then suspended in approximately 50 ml of plasma. This platelet-containing product is then stored until needed for transfusion into a patient.
A number of interrelated factors have been shown to affect platelet viability and function during storage. For example, the anticoagulant used for blood collection, the method used to prepare PC, and the type of storage container used.
The currently accepted standard practice is to store PC for five days at 22.degree. C.; after five days, it has been shown that platelet function may be impaired. In addition to storage time, other storage conditions have been shown to affect platelet metabolism and function including initial pH, storage temperature, total platelet count, plasma volume, and agitation during storage.
One of the major problems in PC storage is regulation of pH. Virtually all units of PC show a decrease in pH from their initial value of approximately 7.0. This decrease is primarily due to the production of lactic acid by platelet glycolysis and to a lesser extent to accumulation of CO.sub.2 from oxidative phosphorylation. As the pH falls, the platelets change shape from discs to spheres. If the pH falls below 6.0, irreversible changes in platelet morphology and physiology render them non-viable after transfusion. An important goal in platelet preservation, therefore, is to prevent this decrease in pH. Platelets must be stored in a container permeable to oxygen since glycolysis is stimulated when oxygen availability is limited.
In association with the decrease in pH, striking deceases in the total amount of ATP per platelet has been observed. It is well known that this reduction of the total ATP level is secondary to the degradation of metabolic ATP to hypoxanthine. The depletion of metabolically available ATP affects platelet function because ATP is essential for such roles in hemostasis as platelet adhesion and platelet aggregation. The ability of PC to maintain total ATP at close to normal levels has been found to be associated with platelet viability.
The composition of platelet storage media has been shown to have a direct effect on the maintenance of platelet function and viability. A number of approaches for the storage of platelets for transfusion have been described.
U.S. Pat. No. 2,786,014 (Tullis) discloses a therapeutic product for injection into humans comprising gelatin, sodium chloride, sodium acetate, carbohydrate (glucose), platelets, and water. It is taught at Col. 2, line 56-62, that the acetate anion acts as an anti-agglutinate for the platelets in this composition. The glucose is disclosed as an example of a hypertonicity-increasing agent.
U.S. Pat. No. Re. 32,874 (Rock et al.) and U.S. Pat. No. 4,447,415 (Rock et al.) disclose a medium for storing platelets in a plasma-free, balanced salt medium. Various additional additives may be added to enhance platelet stability including nutrients, reversible inhibitors of platelet activation, substances to raise cyclic adenosine monophosphate levels, and buffering agents. The disclosed nutrients are fructose, adenine, or acetyl CoA. The reversible inhibitors include indomethacin, quinacrine, or vitamin E. Prostaglandins E1, D2, or I2 are taught for raising AMP levels. The buffering agents disclosed are phosphate or amino acids such as histidine, cysteine, tyrosine, lysine or arginine.
U.S. Pat. No. 4,390,619 (Harmening-Pittiglio) discloses a method of storing and preserving shelf life of platelets for transfusion using ion-exchange resins. These resins provide a source of metabolizing ions in an amount and at a rate sufficient to maintain both pH and ATP levels suitable for transfusion.
While both acetate and phosphate have been used individually in media for storage of platelets for transfusion, the benefits of using them together in a platelet storage medium have not been appreciated previously.